Difference between revisions of "Team:ZJU-China/Design/CNC"

CNC introduction

To make full use of termites’ character, trophallaxis, to eliminate a whole nest of termites, we have to make sure that the worker termites arrive their nest alive. Meanwhile, there should be enough time for them to complete the process of trophallaxis before the toxins function.

Thus, we design bacteria carriers self-assembled from the generated cellulose nanocrystal (CNC).

The function of carriers

To ensure the worker termites’ safe arriving to the nest, the bacteria shouldn’t be digested until specific stimulus comes . Therefore, the bacteria will be embedded in nanomaterial which achieve the controlled release of bacteria by stimuli-center-padness..

A carrier also acts as a protection, preventing the bacteria from being released into environment or simply being degraded without completing its mission.

Thus, the material of the carrier should exsit for a period in termites’ guts but finally be digested by stimuli-center-padness. It also should be dense enough to keep the bacteria from leaking and safe enough to be used in environment.

Using CNC as embedding nanomaterial

CNC is used as the embedding nanomaterial of our genetically modified organism because it meets all demands mentioned.

CNC tends to form multiple hydrogen bonds with the surface of the cell so it achieves self-assembly easily. Termites are sure to eat the embedded bacteria because cellulose is also the main composition of wood, which is the staple food of termites.

Meanwhile, the many attractive features of CNCs, such as their inherent renewability and sustainability, high biodegradability and biocompatibility, high strength, large specific surface area, and nanoscale dimension, have led to effective application[],which provide a proper condition to get bacteria embedded.

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Self-assembly of CNCs and Bacteria

Basing on the interactions between bacteria and the CNCs, we achieve self-assembly in aqueous solution driven by multivalent interactions especially the multiple hydrogen bonds.

Theory and Method

There are abundant binding sites on the surface of bacteria for CNCs. The lipopolysaccharides, form the outer cell membrane of gram-negative bacteria, have plentiful hydroxyl groups to bind with the same functional groups of CNCs by multiple hydrogen bond interactions, while the Van der Waals participate a significant part in polymers as well. As for gram-positive bacteria, the peptidoglycans play an analogous role in the interactions with CNCs. Basing on the properties of the surface of bacteria, we achieve self-assembly between the bacteria and the CNCs driven by multivalent interactions especially the multiple hydrogen bonds.

Bacteria Interaction Assays

E.coli and Streptomycete were separately cultured until an OD600 of 0.5 was attained. The grown bacteria were centrifuged at 12000 rpm for 2 min, and the precipitation was washed in PBS buffer for three times. To E.coli(OD600 = 0.5 A, 1 mL) in PBS, CNC(0.25 mg/mL, 1 mL) was added in a 6-well plate and incubated in an incubator shaker (250 rpm) at 37 °C for 2 h.[]

Generation of the CNCs

Cellulose is the staple food of termites and it has many specific advantages, so we prepare CNC according to the feature of cellulose’s structure. The most used way of producing CNC is TEMPO-mediated oxidation[]. However, we decided to use the way of acidolysis because of its low cost and convenience. In this way, we finally obtained our product of CNC.

Structure of cellulose

Cellulose, is a linear chain of (1-4)-β-D-glucopyranana. Besides the β-(1-4)-glucosidic bond, the intrachain hydrogen bonding between hydroxyl groups and oxygens of the adjoining ring molecules also strengthen the linkage and stabilize the linear structure of the cellulose chain.

In the parallel stacking of multiple cellulose chains, the Van der Waals and intermolecular hydrogen bonds play a significant role, which promote the forming of elementary fibrils that further aggregate into larger microfibrils (5-50 nm in diameter and several microns in length)[].

The multivalent interactions stabilize the cellulose fibrils. Because of the existence of side chains, within these cellulose fibrils there are divided into two kinds of regions where one kind is arranged in a highly ordered (crystalline) structure, and the other kind is disordered (amorphous-like). After acid hydrolysis dissolved the amorphous-like regions, we will get cellulose nanocrystals (CNCs) from the crystalline regions[].

Acid hydrolysis of cellulose

α-cellulose(25 μm，Aladdin) was hydrolyzed at 40 °C with 8.75 mL of 50 wt % sulfuric acid/g of cellulose. To find the most proper time of acidolysis, We carry on a gradient experiment, set up four groups of experiments whose hydrolyzed time are 1h, 2h, 3h and 4h, respectively. The hydrolysis was quenched by diluting 10-fold with cold DI water. []The crystals were collected and washed once by centrifugation for 10 min at 9000 rpm and final solution was collected by centrifugation for 10 min at 5000 rpm. The solution was dialyzed in dialysis bag against ultrapure water until the pH was neutral.

Freeze-drying to get final product

Freeze-drying to get the solid CNC and solve it at 0.25 mg/mL. Crystal aggregates were disrupted by sonicating the suspension for 24 min under ice-bath cooling with a Vibracell ultrasonic processor.